Technical Abstract:
Asian H5N1 highly pathogenic avian influenza has become endemic in several countries, and vaccination is commonly being used. Vaccination can affect surveillance, and therefore there is considerable interest in DIVA (differentiate infected from vaccinated animals) vaccine strategies. Using reverse genetics, we generated a low pathogenic (LP) A/Chicken/Indonesia/7/2003 vaccine virus (rgH5N9) bearing a heterologous subtype N9 NA from an American virus. The rgH5N9 and two North American LP viruses A/Turkey/Wisconsin/68 (H5N9) and A/Chicken/Mexico/232/94 (H5N2) were used as seed strains for inactivated oil emulsified vaccines. Single dose subcutaneous vaccination studies protected chickens from oro-nasal lethal challenge with two Asian HP H5N1 viruses that are phylogenetically in two different lineages, A/Chicken/Indonesia/07/03 (WHO clade 2.1) or A/Chicken/Supuranburi/2/04 viruses (WHO clade 1.0) at 2 weeks post vaccination. The North American viruses differed from the Asian H5N1 viruses by as much as 13% in hemagglutinin amino acid sequence, but still provided 100% clinical protection likely because of the strong antibody responses. The heterologous NA subtype for all three vaccines compared to the challenge strain allowed successful differentiation of vaccinated and infected animals (DIVA) using rapid fluoro-spectrophotometer based neuraminidase antibody inhibition assay.